Dataset Information

Function and Regulation of the Pyruvate Transporter CstA in Escherichia coli.

ABSTRACT: Pyruvate is a central metabolite that connects many metabolic pathways in living organisms. To meet the cellular pyruvate requirements, the enterobacterium Escherichia coli has at least three pyruvate uptake systems-the H⁺/pyruvate symporter BtsT, and two thus far less well-characterized transporters, YhjX and CstA. BtsT and CstA belong to the putative carbon starvation (CstA) family (transporter classification TC# 2.A.114). We have created an E. coli mutant that cannot grow on pyruvate as the sole carbon source and used it to characterize CstA as a pyruvate transporter. Transport studies in intact cells confirmed that CstA is a highly specific pyruvate transporter with moderate affinity and is energized by a proton gradient. When cells of a reporter strain were cultured in complex medium, cstA expression was maximal only in stationary phase. A DNA affinity-capture assay combined with mass spectrometry and an in-vivo reporter assay identified Fis as a repressor of cstA expression, in addition to the known activator cAMP-CRP. The functional characterization and regulation of this second pyruvate uptake system provides valuable information for understanding the complexity of pyruvate sensing and uptake in E. coli.

SUBMITTER: Gasperotti A

PROVIDER: S-EPMC7730263 | biostudies-literature | 2020 Nov

REPOSITORIES: biostudies-literature

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Function and Regulation of the Pyruvate Transporter CstA in Escherichia coli.

Gasperotti Ana A Göing Stephanie S Fajardo-Ruiz Elena E Forné Ignasi I Jung Kirsten K

International journal of molecular sciences 20201128 23

Pyruvate is a central metabolite that connects many metabolic pathways in living organisms. To meet the cellular pyruvate requirements, the enterobacterium Escherichia coli has at least three pyruvate uptake systems-the H+/pyruvate symporter BtsT, and two thus far less well-characterized transporters, YhjX and CstA. BtsT and CstA belong to the putative carbon starvation (CstA) family (transporter classification TC# 2.A.114). We have created an E. coli mutant that cannot ...[more]

PMID: 33260635

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Project description:Pyruvate is an important intermediate of central carbon metabolism and connects a variety of metabolic pathways in Escherichia coli Although the intracellular pyruvate concentration is dynamically altered and tightly balanced during cell growth, the pyruvate transport system remains unclear. Here, we identified a pyruvate transporter in E. coli using high-throughput transposon sequencing. The transposon mutant library (a total of 5 × 105 mutants) was serially grown with a toxic pyruvate analog (3-fluoropyruvate [3FP]) to enrich for transposon mutants lacking pyruvate transport function. A total of 52 candidates were selected on the basis of a stringent enrichment level of transposon insertion frequency in response to 3FP treatment. Subsequently, their pyruvate transporter function was examined by conventional functional assays, such as those measuring growth inhibition by the toxic pyruvate analog and pyruvate uptake activity. The pyruvate transporter system comprises CstA and YbdD, which are known as a peptide transporter and a conserved protein, respectively, whose functions are associated with carbon starvation conditions. In addition to the presence of more than one endogenous pyruvate importer, it has been suggested that the E. coli genome encodes constitutive and inducible pyruvate transporters. Our results demonstrated that CstA and YbdD comprise the constitutive pyruvate transporter system in E. coli, which is consistent with the tentative genomic locus previously suggested and the functional relationship with the extracellular pyruvate sensing system. The identification of this pyruvate transporter system provides valuable genetic information for understanding the complex process of pyruvate metabolism in E. coliIMPORTANCE Pyruvate is an important metabolite as a central node in bacterial metabolism, and its intracellular levels are tightly regulated to maintain its functional roles in highly interconnected metabolic pathways. However, an understanding of the mechanism of how bacterial cells excrete and transport pyruvate remains elusive. Using high-throughput transposon sequencing followed by pyruvate uptake activity testing of the selected candidate genes, we found that a pyruvate transporter system comprising CstA and YbdD, currently annotated as a peptide transporter and a conserved protein, respectively, constitutively transports pyruvate. The identification of the physiological role of the pyruvate transporter system provides valuable genetic information for understanding the complex pyruvate metabolism in Escherichia coli.

Dataset Information

Function and Regulation of the Pyruvate Transporter CstA in Escherichia coli.

Publications

Function and Regulation of the Pyruvate Transporter CstA in <i>Escherichia coli</i>.

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